Microengraving Technology Documents Autoimmune Steps In Diabetes

When trying to observe both the secreted factors and the lineage of certain immune cells, immunologists face what they call their own version of the Heisenberg Uncertainty Principle: an observed immune cell is a perturbed immune cell.

Now, in a proof-of-principle study, scientists have been able to take a much closer look at these cells without disturbing them, using a new microengraving technology. A recent study from the lab of David Hafler, the Jack, Sadie and David Breakstone professor of neurology at Brigham and Women’s Hospital, applied the method to isolate immune cells from clinical samples and print a readout of each cell’s individual characteristics. Their findings suggest that microengraving may become a useful clinical tool for early diagnosis of autoimmune diseases and for monitoring patients’ subtle responses to drugs during clinical trials.

Match-making

The study, described in the October issue of Clinical Immunology, is the result of a serendipitous connection between Elizabeth Bradshaw, first author and a research fellow in Hafler’s lab, and microengraving in-ventor J. Christopher Love, now an assistant professor of chemical engineering at MIT.

Love developed the microengraving method in 2006 at HMS as a research fellow in the lab of Hidde Ploegh. He designed the technology to isolate cells that produce specific antibodies so these cells could be retrieved and cloned.

Meanwhile, Bradshaw had begun a project to examine cells collected from the pancreatic lymph nodes of deceased diabetic patients. She focused on B cells that produce antibodies against insulin. She wanted to see if all of the insulin-reactive B cells had the same genes. Shared genes imply that the cells are the result of a clonal expansion in which one cell clones itself after detecting an antigen.

She began sequencing the B cell receptors in an effort to find the autoreactive cells. But after sequencing cells from three human samples, she stopped.

“The problem with working with the lymph nodes is that the background is very high,” said Bradshaw. “I was sequencing hundreds of cells trying to find an antigen-reactive B cell. It wasn’t working.”

A Versatile Technique

Shortly after she decided that she needed to reevaluate her approach, Bradshaw attended a workshop where she saw Ploegh, now a professor of biology at MIT, present the microengraving method. “I thought, oh my God, that is the technique that is going to let me ask my question,” she said. The idea meshed with longstanding conversations between Hafler and Ploegh, who together helped Bradshaw forge a connection with Love.

The microengraving technology is based on intaglio printing, an etching technique invented in the 1430s. Love adapted the copper-plate–based method to use microscale wells that are molded using soft lithography.

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To use the tool, scientists flow the cells they want to study over the wells, depositing one or two cells per well. In the case of the experimental B cells, Bradshaw stimulated them first and deposited them in the wells. She then serially inverted the wells onto plates coated with antibodies tuned to capture certain secretions.

The inverted wells imprint the cells’ secreted factors onto the plate like a stamp; the secretions stick to the coating like ink to paper. The plate is then coated with fluorescently labeled antibodies—analogous to pigment—to label each different secreted factor with a different color.

“The beauty of this technology is that it is very simple and very flexible. You can put down primary circulating lymphocytes; you can put down isolated cell types; you can do just B cells, just T cells, so as a technique it allows us a lot of freedom to apply it to whatever we want,” said Bradshaw.

It could, for example, be applied to allergies, said senior author Hafler. Because microengraving allows clinicians to look at the antigen specificity of an immune cell as well as the factors it secretes, it may help scientists better un-derstand which specific cells are responsible for causing an allergic reaction.

To the Clinic

In this first proof-of-principle clinical application, which focused on diabetes, Bradshaw applied the technology to two patient blood samples. She obtained one sample from a patient with type 1 diabetes and high levels of autoantibodies against insulin in the bloodstream. These high titers suggested that the patient might have autoreactive B cells tuned to detect and capture insulin. She obtained the second sample from a healthy control.

Bradshaw brought the samples to Love’s lab, where the microengraving system, still a manual prototype, is set up. Using proinsulin as one coating, the researchers found that the diabetic patient did, indeed, have B cells secreting insulin autoantibodies. In addition, these same B cells secreted IgG rather than IgM. This demonstrated that the B cells had undergone a switch in response to some antigen.

Bradshaw now plans to ask her original question: did all of these switched insulin-reactive B cells come from the same cell in one clonal expansion? To answer this, she can retrieve these rare B cells out of the microwells and sequence them, knowing that each time she will be sequencing a cell of interest.

“The ability to interrogate individual cells not only for function—by what they secrete or what they do—but also in terms of their RNA and their receptors, is to me a real breakthrough,” said Hafler.

The group is now doing the sequencing, but it is unproven territory. The lab has successfully analyzed T cells in the wells and then retrieved them for cloning, but they have not yet done it for B cells. As with this entire study, applying the new technology involves trial and error to find a process that works and is highly reproducible.

Love’s lab is continuing to refine the technology to make it faster to use. He also would like to expand its capabilities so it extracts more information from each cell and provides a fuller picture of what is present in each sample, he said.

“Hopefully this technique will eventually become a kit,” said Bradshaw. “You can buy it, do the tests, and get a printout of your 50 clinical samples the next day.” - Elizabeth Dougherty

Comments

The Tuberculosis Test is based on intaglio printing, an etching technique invented in the 1430s. Love adapted the copper-plate–based method to use microscale wells that are molded using soft lithography.

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